Peptides antagonistic to an anti-angiogenic antibody and uses therefor

ABSTRACT

The invention concerns antagonists of alphav-containing integrins which have therapeutic activity in oncology applications and methods for selecting such antagonists using a peptide which competes for alphaV-integrin binding with a known monoclonal antibody having demonstrated anti-tumor activity. The claimed peptide represents a conformational epitope or mimotope present on the ligand to which the therapeutic antibodies selectively bind.

BACKGROUND OF THE INVENTION CROSS REFERENCE TO RELATED APPLICATION

This application is a non-provisional application filed under 37 CFR1.53(b)(1), claiming priority under 35 USC 119(e) to provisionalapplication No. 60/480,667 filed Jun. 23, 2003, the contents of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods of characterizing therapeuticantibodies and using information about binding specificity and moreparticularly as defined by a unique peptide ligand. The inventionrelates to therapeutic proteins which interact with alphaV containingintegrin receptors.

BACKGROUND OF THE INVENTION

Antibodies and T-cell receptor molecules possess variable regions thatare responsible for specific antigenic recognition. The region of theantigen that is bound by the antibody or T-cell receptor is termed theantigenic determinant or epitope. Similarly, the variable regions ofantibodies and T-cell receptors also contain determinants, or“idiotypes” that are immunogenic and are capable of initiating ananti-antibody response, an anti-idiotype (“anti-id”) immune response.

More particularly, idiotopes are associated with the variable regions ofantibodies and T-cell receptors. These variable regions confer antibodyand T-cell receptor specificity for antigens. Idiotypes are immunesystem markers on antibodies and T-cell receptors. An idiotype isimmunologically defined by reactivity with more than one anti-idiotypicantibody that recognizes an idiotypic determinant or idiotope within agiven idiotype; therefore, an idiotype is made up of a collection ofidiotopes.

Idiotopes are best defined by their binding to monoclonal anti-idiotypicantibodies. It also should be noted that idiotopes are distinct fromisotypic (immunoglobulin class-specific), xenotypic (species specific)and allo-typic (certain population sub-group specific) determinants.

Each antibody and T-cell receptor has at least one paratope that is thebinding site for an antigen determinant (the epitope). A paratope mayserve as an idiotope, that is, the paratope may stimulate ananti-idiotypic response in which, like the original antigen, ananti-anti-idiotopic antibody binds to an epitope within the paratope. Asubset of anti-paratope anti-idiotype (“anti-id”) antibodies may mimicthe immunologic properties of the original antigen and are known as“internal image” antibodies. In addition to the anti-paratopic anti-idsthat mimic the original antigen, other anti-id antibodies defineantibody and T-cell receptor idiotopes that also participate in theregulation of immune responses. These idiotypes are termed regulatoryidiotypes and they are not necessarily “internal images” of the originalantigen. For a general discussion of these background principles, seeBurdette, S. and Schwartz, R., New Eng. J. of Med. 317:219 (1987).

Epitope mapping a monoclonal antibody or, in some cases, polyclonalserum, is generally understood to mean the process of deducing the exactregion of the antigen or target molecule for which the antibodypreparation has the highest affinity. A determinant or epitope of atarget generally is understood to mean the portion of an antigen towhich the most robust immune response is generated in terms of avidityand selectivity of binding. Peptides or other small fragments of anantigen can be used as immunogen (Niman et al. Proc. Natl. Acad. Sci.USA 1983, 80:4949-4951 and U.S. Pat. No. 5,030,565). In some casespeptides having unrelated sequences or structures can behave as epitopesor, mimotopes, in terms of being able to act as a binding partner for anantibody or compete for binding with the original antigen. Sequenceanalysis of these peptides can lead to identification of the structuraland physicochemical features of the epitope. Systematic methods ofaltering peptide sequence and testing for antibody binding orcompetition between antigen and peptide have been used to performepitope mapping. One such method, taught by Geysen et al. (Proc. Natl.Acad. Sci. USA 81: 3998-4001, 1984 and WO8403564) became widely used asit coupled the “pin” technology for solid phase peptide synthesis toligand binding assays (sold as the Multipin Peptide Technology (PepScan)by Chiron Mimotopes, San Diego, Calif.). In numerous cases, theseepitopic peptides have little or no sequence homology with the originalantigenic protein or peptide (Geysen et al. Mol. Immunol. 23: 709-715,1986). This finding lent support to the concept that, in some cases,antibodies recognized “conformational epitopes” which are only formed inthree-dimensional space upon folding and twisting of the linear sequenceor because of association with another peptide as in heteromultimers.Conformational epitopes are also termed mimotopes. WO8600991A1 andWO8606487A1 teach methods of determining conformational epitopes.

Another widely used method for random generation of libraries of diversepeptides for studies is the use of bacteriophage expression systems orphage display. Phage display technology provides the additionaladvantage that the peptide or protein displayed on the coat protein of abacteriophage is physically linked to its genetic constituents withinthe phage particle (Smith, Science 228: 1315-1317,1985). Such librarieshave been used as the molecular biological equivalent of the Geysonmethod (Devlin et al. Science 1990, 249:404-406; Pluckthun, A. Curr. Op.Biotech. 1991, 2:238-246).

Therefore, the process of epitope mapping provides information about anantigenic molecule, which when linked to information about thebiological activity of the antigen or properties altered in the presenceof an antibody, provide a means to deduce or understand the biologicalfunctions of the target represented by an antigen or epitope andconversely the scope of the possible effects of an antagonistic antibodywhich prevents normal interaction of that epitope with its naturallyoccurring cognate ligands.

Directed biopharmaceutic drug design is highly desirable. Therefore, anunderstanding of protein-protein interactions and conformationspecificity in target-substrate and target-ligand interactions isimportant. U.S. Pat. No. 6,143,876 teaches a method of obtainingantibodies to a specific epitopes of a complex which is only revealedupon formation of that complex. WO0170984 describes in detail thesignificance of understanding the surface epitope bound by a Mabspecific for a protein involved in coagulation as the interaction withthis factor, called tissue factor, which interacts with multiple ligandssequentially.

Integrins are a family of heterodimeric transmembrane receptors thatmediate cell-cell and cell-extracellular matrix adhesion. Signaltransduction by integrins has been shown to regulate diversifiedfunctions, including cell differentiation, proliferation, migration,apoptosis and angiogenesis (Shimizu et al., Advances in Immunology 72:325-385,1999; Hynes, Cell 69: 11-25,1992). Alpha-V integrins comprise asubset sharing a common alpha-V subunit combined with 1 of 5 betasubunits (beta-1, -3, -5, -6, or -8. All or most alpha-V integrinsrecognize the sequence RGD in a variety of ligands (vitronectin,fibronectin, osteopontin, bone sialoprotein, thrombospondin, fibrinogen,von Willebrand factor, tenascin, and agrin) and, in the case ofalphaVbeta8, laminin and type IV collagen. Blocking or inhibiting theprocesses associated with integrin signaling is therefore a logicalapproach to preventing, treating or limiting the spread of cancer in thebody and other diseases such as those of the eye and cardiovascularsystem characterized by inappropriate angiogenesis or cellular adhesionand proliferation. Accordingly, applicants have discovered a monoclonalantibody that binds and blocks the activation of alphaV containingintegrin receptors, which antibody is disclosed in copending applicationpublished as WO0212501.

Given the above mentioned value that epitope mapping provides, it can beseen that the discovery of any cognate ligand for a therapeutic antibodyprovides not only a novel mimotope that can function as an alternatedirected antigen but further provide a significant tool to measureamount and function of that therapeutic antibody. The identification ofligands to CNTO95 mAb, by identifying antagonist peptides that caninhibit binding of CNTO95 to integrins αVβ3 and αVβ5, is one strategyfor the discovery of such a composition.

SUMMARY OF THE INVENTION

The invention relates to a peptide of the formula:

Asp Phe Xaa₁ Ser Xaa₂ Trp Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈, wherein Xaa₂ isselected from Trp, Tyr and Phe, Xaa₃ is selected from Glu and Asp, Xaa₄is selected Ile, Leu and Val and Xaa₁, Xaa₅ Xaa₆ Xaa₇ Xaa₈ areindependently selected from any naturally occurring amino acid (SEQ. ID.NO: 1);

-   -   which peptide is a high affinity antagonist of an anti-alphaV        integrin heterodimer antibody. More particularly, the peptide of        the invention is a mimotope of CNTO95.

In one embodiment, the invention relates to a linear 12-mer peptide ofthe formula DFRSWWDLSGYR (SEQ. ID. NO: 2).

The peptides of the invention may be used as a method of therapeuticallyvaccinating a patient suffering from a disease characterized byangiogenic and metastatic processes.

The peptides of the invention may be used to immunize an animal for thepurpose of generating a therapeutic antibody capable of treating orpreventing diseases characterized by angiogenic and metastaticprocesses.

The peptides of the invention may be used in a method of screening fortherapeutic or diagnostic antibodies that recognize a mimotope based ona structure formed in alphaV containing integrin receptors.

The peptides of the invention may be used in a method of selectingtherapeutic anti-integrin molecules that bind the CNTO95 mimotope basedon a structure formed in alphaV containing integrin receptors.

In another aspect, the invention relates to an isolated anti-integrinantibody which specifically binds at least one epitope comprising one ormore, preferably 3 or more, of the amino acids of the peptide of SEQ.ID. NO: 1, other than the antibody designated CNTO 95 disclosed inpublished patent application WO0212501. In accordance with theinvention, such antibody has at least one anti-integrin activity,including but not limited to inhibition of vitronectin binding,inhibition of binding of alpha V to at least one alpha V receptor orligand, angiogenesis modulation or binding to integrin expressing cells.An antibody according to the invention includes any protein or peptidecontaining molecule that comprises at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that can be incorporated into anantibody of the present invention.

An antibody of the invention can include or be derived from any mammal,such as but not limited to a human, a mouse, a rabbit, a rat, a rodent,a primate, or any combination thereof and includes isolated human,primate, rodent, mammalian, chimeric, humanized and/or CDR-graftedanti-integrin antibodies, immunoglobulins, cleavage products and otherspecified portions and variants thereof. The invention also relates toanti-integrin antibody compositions, encoding or complementary nucleicacids, vectors, host cells, compositions, formulations, devices,transgenic animals, transgenic plants, and methods of making and usingthereof, as described herein together as combined with what is known inthe art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a graphic representation of the crystal structure of alphaVshowing the position of D564-Q575 segment of the mature chain betweenthe b-propeller and thigh structures.

FIG. 2 is a graphic representation of the 3-dimensional spatialpositioning of the D564-Q575 segment of the mature chain.

FIG. 3A-C is a multiple alignment generated using ClustalW sequence ofhuman alphaV, alpha2B, and alpha5 (GenBank Accession Nos: NP_(—)002201,A34269, and NP_(—)002196, respectively) with the D564-Q575 segment ofthe mature chain marked.

FIG. 4 contains graphs showing the results of ELISA assays demonstratingthe concentration dependence of CNTO95 mAb binding to syntheticpeptide-coated microwell plates: CEN2319 (mimotipe) and CEN2322 (alphaVsegment) (A); direct comparison of CNTO95 binding, an anti-alphaVbeta3Mab (LM609) and an anti alphaVbeta5 Mab (PIF6) to CEN2319 coated plates(B); and results of ELISAs for CNTO1275 mAb binding to syntheticpeptides (C).

FIG. 5. contains graphs showing the results of ELISA assaysdemonstrating the concentration dependence of synthetic peptides bindingto microtiter plates coated with alphaVbeta3 (A); and a graph showingthe results of ELISA assays demonstrating the concentration dependenceof synthetic peptides binding to microtiter plates coated withalphaVbeta5 (B).

FIG. 6 are tracings and graphs showing the titration calorimetry datafor CEN2319 peptide binding to CNTO95 mAb (A); and titration calorimetrydata for CEN2322 peptide binding to CNTO95 mAb (B).

FIG. 7 is a graph showing the affect of CEN2319, CEN2553, the nativepeptide CEN 2322, on the inhibition of cellular adhesion by CNTO95 tovitronectin coated plates.

DETAILED DESCRIPTION OF THE INVENTION

Phage-displayed peptide libraries were used to identify novel ligands tothe human monoclonal antibody, CNTO95. CNTO95 is a fully human IgG1,kappa monoclonal antibody produced at Centocor from a GenPharmtransgenic mouse and has been shown to recognize integrins αVβ3 and αVβ5in an EDTA-insensitive manner The CNTO95 anti-integrin antibody isdisclosed in patent application WO0212501, hereby incorporated byreference into the present application.

Phage-displayed peptide libraries were probed in order to identify thosepeptides capable of preventing binding of Centocor monoclonal antibody,CNTO95, to its ligands, integrins αVβ3 and αVβ5. Screening of librariesof random 7-mers, 12-mers and cyclic 9-mers against CNTO95 identifiedsuch antagonist peptides. A linear 12-mer peptide, with the sequenceDFRSWWDLSGYR (CEN2319), inhibits the interaction between CNTO95 and bothintegrin αVβ3 and αVβ5. Synthetic linear peptide CEN2319 was analyzedfor CNTO95 binding activity using competitive enzyme-linkedimmunosorbent assays. The CNTO95 binding affinity of peptide CEN2319 wasdetermined by this assay, giving EC50 value of 5.8 μM. From titrationmicrocalorimetry analysis, peptide CEN2319 was shown to bind the CNTO95with 2:1 stoichiometry and its binding affinity was 4.2 μM.

The biophysical properties of CEN2319 indicate that identification ofsmall peptides presenting binding capacity to the monoclonal antibodyCNTO95 are feasible and that such peptides can guide the discovery ofother therapeutic agents specific for alphav-containing integrins. Thispeptide, which represents an antibody mimotope is a useful lead for theselecting of integrin receptor antagonist antibodies and peptides. Inaddition, these studies suggest strategies for mimicking binding siteson integrin receptors with phage displayed peptides.

The biophysical properties of CEN2319 as more generally indicated by theformula represented by SEQ. ID NO: 1 can be used to create otherpeptides which may, due to biological or chemical properties beadvantageously used for the purposes of the invention. The substitutionof amino acids of like properties is well known in the art and is termedconservative substitution. The twenty naturally occurring amino acidshave been variously categorized into groups. These groups include aminoacids with basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar orhydrophilic side chains (e.g., glycine, asparagine, glutamine, serine,threonine, tyrosine, cysteine), non-polar or hydrophobic side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

As stated above, in one aspect of the present invention, there is to anisolated anti-integrin antibody which specifically binds at least oneepitope comprising one or more, preferably 3 or more, of the amino acidsof the peptide of SEQ. ID. NO: 1, other than the antibody designatedCNTO 95 disclosed in published patent application WO0212501. Theantibody preferably further binds an alpha V integrin with an affinity(K_(d)) of at least 10⁻⁹ M, preferably at least 10⁻¹⁰ M, and/orsubstantially neutralize at least one activity of at least one alpha Vintegrin protein. In a preferred embodiment, the antibody binds an alphaV integrin protein with an affinity (K_(d)) of at least 1×10⁻¹¹ M,preferably 5×10⁻¹¹ M.

The present invention further provides, in one aspect, isolated nucleicacid molecules comprising, complementary, or hybridizing to, apolynucleotide encoding the aforementioned specific peptides orantibodies thereto, comprising at least one specified sequence, domain,portion or variant thereof. The present invention further providesrecombinant vectors comprising said peptide or antibody encoding nucleicacid molecules, host cells containing such nucleic acids and/orrecombinant vectors, as well as methods of making and/or using suchnucleic acids, vectors and/or host cells. Thus, the invention comprisesisolated nucleic acid encoding at least one isolated peptide of Seq. IDNo. 1 or mammalian anti-integrin antibody which binds such peptide; anisolated nucleic acid vector comprising the isolated nucleic acid,and/or a prokaryotic or eukaryotic host cell comprising the isolatednucleic acid. The host cell can optionally be at least one selected fromE. Coli, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0,293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or anyderivative, immortalized or transformed cell thereof. Also provided is amethod for producing at least one peptide or antibody of the invention,comprising translating the peptide or antibody encoding nucleic acidunder conditions in vitro, in vivo or in situ, such that the peptide orantibody is expressed in detectable or recoverable amounts.

The antibodies of this invention can be prepared in several ways wellknown in the art. In one aspect, the antibodies are convenientlyobtained from hybridomas prepared by immunizing a mouse with thepeptides of the invention. The antibodies can thus be obtained using anyof the hybridoma techniques well known in the art, see, e.g., Ausubel,et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001),each entirely incorporated herein by reference.

Abbreviations

-   -   Abs antibodies, polyclonal or monoclonal    -   aV integrin subunit alpha V    -   b3 integrin subunit beta 3    -   b5 integrin subunit beta 5    -   Ig immunoglobulin    -   IgG immunoglobulin G    -   Mab monoclonal antibody.

While having described the invention in general terms, the embodimentsof the invention will be further disclosed in the following examples.

EXAMPLE 1 Affinity Selection of Three Phage-Displayed Peptide Librarieson CNTO95

Affinity selection of phage-displayed peptide libraries was carried out.Three libraries, linear 7-mer, linear 12-mer and cyclic 9-mer, have beenscreened against CNTO95. One hundred μl of each phage library, at aconcentration of about 5×10¹⁰ pfu/ml in TBST buffer, was incubated in amicrotiter well which was previously coated with 0.1 mg/ml of CNTO95 in0.1M NaHCO₃ (pH 8.6) buffer. After incubation with constant rotation for2 hours at 37° C. to facilitate the binding interaction, the wells werewashed 10× with TBST buffer. The remaining bound phage were eluted with100 μl of 0.1 mg/ml CNTO95, or 0.2 M Glycine-HCl (pH 2.2) andneutralized with 25 μl of 1 M Tris-base (pH 8.0). The eluted phages werepropagated for use in the next affinity selection cycle. After threerounds of selection, an aliquot of eluted phage solution was plated ontoa lawn of permissive E. coli ER2738 agar plates and colonies allowed togrow overnight at 37° C. Approximately 20 individual colonies wererandomly selected and the sequence of the inserted peptide ligand wasdetermined by DNA sequencing. The amino acid sequences of thephage-displayed peptides isolated by panning against CNTO95 are shown inTable 1. TABLE 1 (eluted with 0.2 M Glycine-HCl, (eluted with 100 μg/mlCNTO95) pH 2.2) Peptide Sequence Frequency* Peptide Sequence Frequencylinear 12-mer 12p1 DFRSWWDLSGYR 14 (28) 12pG1 DFRSWWDLSGYR 7 (8) 12p2SKFQSYWELFPY  8 (28) 12pG2 TH PNL SHAAQTR 1 (8) 12p3 MPHKHEIWDWWY  5(28) 12p4 STHLLPKPIMTN  1 (28) linear 7-mer 7p1 ALGHSFP  9 (17) 7pG1HSVYYPV 5 (10) 7p2 SMERPFV  4 (17) 7pG2 SHPASHD 2 (10) 7p3 LPLTPLP  2(17) 7pG3 SMPPGLP 1 (10) 7p4 MLTPPNP  1 (17) 7pG4 ALGHSFP 1 (10) 7p5STTTAPR  1 (17) 7pG5 SFVLPYY 1 (10) cyclic 9-mer C7Cp1 CSPLFTPWC 17 (17)C7CpG1 SPLFKPW 9 (12) C7CpG2 ESHSRPH 2 (12) C7CpG3 PQSEMDR 1 (12)*Numbers in parentheses refer to the total number of plaques sequenced.

Within linear 12-mer group, there was an apparent preference (>58%) forthe sequence of DFRSWWDLSGYR. Within the cyclic-9 mer binders to CNTO95,the sequence of SPLFXPW (SEQ. ID. NO: 3) dominated in the randomlyselected clones.

The linear 12-mer consensus peptide sequences has been aligned withsequences of the human integrin alpha subunits V, as well as betasubunit 3. The sequence alignment results were shown in Table 2, wherematches are underlined with a solid line for exact identities andconservative substitutions with a dotted line. TABLE 2 Human alphaV₅₆₁YRLDYRTAADTTGLQPILNQ₅₈₀ Linear 12-mer       DFRSWWDLSGYR Cyclic 9-mer       SPLFXPW Human beta3 ₁₂₉VRQVBDYPVDIYYLMDLSYSMKDDL₁₅₃ Linear 12-mer            DFRSWWDLSGYR cyclic 9-mer              SPLFXPW

After comparing the peptide sequences to the extracellular segments ofintegrin αVβ3, it was found that among the possible matches or regionswith close homology the 12 residue segment D564-Q575 of the mature chainof αV was the closest match of surface-exposed segments. There waslittle or no homology to any surface exposed region of the betasubunits. This analysis was based on the crystal structure of a solubleform of αVβ3 published by Xiong et al. (Science 294: 339-345, 2001 andScience 296: 151-155, 2002) shown in FIG. 1. The region forms a loopprotruding from the Thigh (439-592) domain towards the beta-propeller(1-438) (FIG. 2). A multiple sequence alignment of alphaII, alpha5, andalphaV show this to be the region of least homology between the species,indicating that this domain is unique to the alphaV integrin family anda good candidate for a subunit specific epitope (FIG. 3B).

Based on this analysis, two synthetic peptides, CEN 2319 (DFRSWWDLSGYR,consensus sequence selected out from linear 12-mer peptide library) andCEN2322 (₅₆₄DYRTA ADTTGLQ₅₇₅, sequence of αV) (SEQ. ID. NO: 4) weresynthesized and examined for their binding affinities by competitionELISA for further validation. The peptide, SPLFXPW (SEQ. ID. NO: 3flanked by cysteine residues which form a disulfide bond), was alsotested for binding to CNTO95 using the ELISA format but no specificbinding was detected.

EXAMPLE 2 Elisa for CNTO95 Binding to Synthetic Peptides

To verify the binding activity of synthetic peptides to CNTO95 mAb,ELISAs were performed using peptide-coated plates. PRO-Bind ELISA plates(Falcon) were coated with 100 μl (100 μg/ml) of either CEN 2319, CEN2322or PBS buffer. Peptide-charged ELISA plates were incubated with 100 μlof various concentrations of CNTO95 mAb for 2 hours at 37° C., and thewells then were washed with PBS plus 0.5% Tween 20 (PBST). Binding wasdetected with a 1:5,000 dilution of of peroxidase-conjugated goatanti-human IgG, Fc gamma fragment antibody (Jackson) and colordevelopment with 3,3′,5,5′-tetramethyl-benzidine dihydrochloride (TMB).The reaction was detected by reading the absorbance at 450 nm.PBS-coated wells were used as background control. The results showedthat synthetic peptide CEN2319 could be recognized specifically by mAbCNTO95 (FIG. 4A). In contrast, commercially available antibodies toalphaVbeta3, LM609 (Chemicon, Temacula, Calif.) and to alphaVbeta5, PIF6(GIBCO, Gaithersburg, Md.) did not bind the CEN2319 coated plates (FIG.4B). Another set of negative control was performed using mAb CNTO1275(FIG. 4C). The binding signal is negative.

Conclusion: CEN2319 indeed shows specific binding to CNTO95 (FIG. 4A)but not to other antibodies recognizing alphaV-containing integrins(FIG. 4B) and not to another human mAb to an unrelated antigen, CNTO1275(FIG. 4C). The non-binding antibodies, clone LM609 and P1F6, have beenpreviously shown not to compete with CNTO95 (WO0212501). Therefore, thepeptide CEN2319 binding assays support that the epitope bound by CNTO95is distinct from other integrin binding Mabs. A peptide (CEN2322) madefrom an actual sequence segment of alphaV also did not bind to CNTO95(FIG. 4B) indicating that a unique non-linear epitope is recognized byCNTO95.

EXAMPLE 3 Evaluation of Binding Specificity for CEN2319 Peptide toCNTO95

Competitive ELISA on microtiter plates coated with integrins αVβ3 orαVβ5 were carried out to evaluate the binding specificity for CEN2319peptide to CNTO95 mAb. 100 ul of 5 μg/ml either αVβ3 or αVβ5 (Chemicon,Temacula, Calif.) was coated on the ELISA plates at 40° C. over night.Then, 100 μl of 2 μg/ml CNTO95 was pre-incubated at room temperature for30 minutes with various concentration peptides (100 μl), ranging from 0to 200 μg/ml. The peptide-antibody mixtures were then added to theintegrin-coated microwells. Following incubation at 37° C. for 2 hours,the plates were washed thoroughly, and bound CNTO95 was detected usingperoxidase-conjugated goat anti-human IgG, Fc gamma fragment antibody(1:5,000 dilution), followed by substrate addition as above. EC50 valuewas determined by the concentration of competing synthetic peptide thatresulted in half-maximal CNTO95 binding to integrins αVβ3 or αVβ5.

CEN2319 and CEN 2322 synthetic peptides were analyzed for bindingspecificity using competitive ELISA on plates coated with variousintegrins. The data, shown in FIGS. 5A and 5B were used to calculate theEC50 for CEN2319 peptide to compete CNTO95 mAb binding to aVb3 and whichwas determined to be 5.8 μM (FIG. 5A). On αVβ5-coated plates, CEN2319alone showed significant inhibition of CNTO95 binding to integrin αVβ5.CEN2322 peptide did not compete with CNTO95 binding to αVβ3 or αVβ5.

Overall, the competitive ELISA data demonstrated that synthetic peptideCEN2319 inhibits CNTO95 mAb binding to integrins αVβ3 and αVβ5.

EXAMPLE 4 Binding Affinities and Stoichiometries of Synthetic Peptidesfor CNTO95

Titration microcalorimetry is a technique used to measure equilibriumbinding affinity by monitoring the enthalpy of binding as a function oftitrant added to second molecule at fixed temperature. To determinebinding affinities and stoichiometries of synthetic peptides for CNTO95,titration calorimetry experiments were carried out with a Microcal(Amherst, Mass.) MCS isothermal titration calorimeter. Proteinconcentrations were measured by absorbance at 280 nm. Aliquots ofpeptide were titrated into a fixed concentration of CNTO95 at 3 μM inDulbecco's PBS. Heats of binding are monitored with each injection.Peptides were added until the observed heats approach the heats ofdiluted peptides into buffer. Calorimetry data were analyzed withMicrocal ORIGIN software according to a single-site binding model ineach case (Wiseman et al., Anal. Biochem. 179: 131-137,1989). The modelincludes an equilibrium binding constant (Kd), a molar binding enthalpychange (ΔH) and a molar binding ratio for the binding reaction at 25° C.Table 3 gives the calculated CNTO95 binding properties of syntheticpeptides from titration calorimetry data. TABLE 3 Molar ratio ( ΔH KdPeptide Peptide/CNTO95) (cal/mole) (μM) Comments CEN2319 2.2 −12,000 4.2CEN2322 — −325 — No binding

In this study, we have demonstrated that two synthetic CEN2319 peptidesbind to one CNTO95 mAb and the binding affinity for CEN2319 peptidegiven as Kd value is 4.2 μM (FIG. 6A and Table 3).

EXAMPLE 5 Positional Substitution Analysis of Peptide CEN2319

In order to further understand the contribution of each amino acidresidue in the binding, systematic substitution at each position weremade and tested. The binding analysis was carried out by spotting thevarious peptides on a nitrocellulose membrane and probing with CNTO95followed by HRP-conjugated goat-anti-human Fc antibody and colordeveloped using a peroxidase substrate. The relative densities of colorat each spot were then scored as given in Table 4 below. TABLE 4Position 1 2 3 4 5 6 7 8 9 10 11 12 Original D F R S W W D L S G Y RResidue A +++ ++ + ++ +++ R + ± ± + N + +++ + + ++ D + ++ ++ ± ++ ++ +++C Q ++ ± ++ ++ ++ ++ E + +++ ± ++ +++ +++ +++ G ± ± + ++ ++ H ± ± ± ± ±± ± ++ ++ ++ I ± ++ + ± + ++ L +++ ++ ± ± + ++ K ± ± M ++ ± +++ + + +++F + ++ ++ ± ++ + ± + P ± ++ ++ ++ S +++ + + + + ++ T + ± ++ + ++ ++ W ±++ + ± + + + Y ± + +++ ± +++ + + + V ± ++ ++ + ++ ++ Allowed D F X S Y/W E/D I/L/V X X X X Residues W/FIntensity:+++: very strong;++: strong;+ equivalent;±: weak;Blank: negative.

These data show that the allowable positional substitutions are: anaromatic residue at position 5, a negative charge at position 7, and ahydrophobic residue at position 8. Substitutions at position 3 and 9-12had no substantial effect, while any substitution at positions 1,2,4 and6 dramatically reduced the binding of CNTO95 to the peptide.

EXAMPLE 6 Preparation of an Alternate Mimetope Peptide

Using the derived consensus formula (SEQ. ID. NO: 1):

-   -   DFXS----(Y/W/F)----W---(E/D)--------(I/L/V)----------X₄    -   DFXS---Aromatic----W-Negative charge--Hydrophobic-Optional        Branched Chain    -   an alternate antagonist peptide, CEN2553, having the sequence        DFRSWWDLEE, (SEQ ID. NO: 5) was synthesized and tested for the        ability to prevent CNTO95 from binding its target on the cell        surface and by a functional assay. The C-terminal glutamate        residues were added to increase overall solubility of the        peptide only.

For the detection of surface integrins, A375.S2 cells (human melanomacells) were harvested, rinsed, suspended in unsupplemented RPMI media.The antibody, CNTO95, was incubated at a final concentration of 10 ug/mlwith or without peptides at the concentrations given in Table 1 for 1hr. Then, the cell solution was added and incubated for an additonal 60minutes on ice followed by addition of PE-labeled goat anti-humanantibody IgG-Fc (10 ug/ml). Absence of primary antibody or substitutionof primary antibody with isotype matched antibody served as negativecontrols. Cells were immediately analyzed with a FACS Scan II flowcytometer (Becton Dickinson, Mountain View, Calif.). The data wasplotted as counts versus PE fluorescence and showed, in each case, asingle distinct peak. For simplicity, the mean channel fluorescence foreach peak was used as the relative staining value (Table 5). The resultsshowed that competitive peptides selected from phage display and amodification based on the positional substitution analysis bothinhibited CNTO 95 binding to cells. TABLE 5 Conc of Peptide Mean ChannelPEPTIDE (ug/ml) Fluorescence Cells Only — 3.36 Cells + CNTO95 only —89.7 +CEN2319 100 12.27 +CEN2319 200 11.60 +CEN2322 100 90.55 +CEN2322200 92.40 +CEN2553 100 12.95 +CEN2553 200 9.41

For cell adhesion assays, Microtiter plates (Linbro-Titertek, ICNBiomedicals, Inc) were coated at 4° C. overnight with vitronectin (1mg/ml). Immediately before use plates were rinsed with PBS and blockedfor 1 hour with 1% BSA/PBS (pH 7.4). Adherent cells (M21, human melanomacells) were labeled with Calcein AM fluorescent dye (Molecular Probes,Eugene, Oreg.) according to the manufacturer's instructions, harvested,washed twice, and suspended in 0.1% BSA in DMEM medium. Variousconcentration of Antibodies were incubated with peptides for 1 hrs. Celldensity was adjusted to 5×10⁵/ml and they were incubated with antibodyand peptide solution for 15 min at 37° C. The cell-antibody mixture wasadded to wells (100 μl per well) and incubated for 1 h at 37° C. Plateswere rinsed twice with PBS to remove unbound cells and adhesion wasmeasured in a fluorescence plate reader (Fluoroskan) at 485-538 nm. Celladhesion to BSA-coated wells served as a negative control. Isotypematched antibodies served as a negative control. FIG. 7 shows that theeffect of the 10-mer sequence (SEQ. ID. NO: 5) based on the derivedformula (SEQ. ID. NO: 1) was indistinguishable from the peptide CEN2319(SEQ. ID. NO: 2) isolated originally by capture of phage displaying thepeptide by binding to CNTO95 directly.

SUMMARY

The equilibrium affinity Kd value, 4.2 μM, for CEN2319 peptide bindingto CNTO95 was obtained by titration microcalorimetry (FIG. 6A and Table3). There is no evidence for CEN2322 peptide binding to CNTO95 mAb (FIG.6B and Table 3). These data were in good agreement with those obtainedfrom competitive ELISA data (FIGS. 5A and 5B). Evidence for 2:1stoichiometry for CEN2319 peptide to CNTO95 mAb using titrationmicrocalorimetry (FIG. 6A) was also obtained. Molar binding ratio wasdetermined by non-linear least squares analysis of the data in FIG. 6Aand was given in Table 3. Deviation from theoretical 2.0 are likely dueto the inherent errors in defining reactant concentrations by absorbanceat 280 nm and weighing the mass of synthetic peptides. In this case, themolar binding ratio 2:1 for CEN2319 peptide to CNTO95 argues stronglythat each Fab arm of CNTO95 mAb binds one peptide.

Using data from alignments of the binders and as compared to the parentsequence of the human alpha V integrin subunit, a formula was derived,which maps subregions of the binding surface necessary for thepreparation of an antagonist peptide to the Mab CNTO95. The utility thisformula was demonstrated as a peptide (SEQ. ID. NO. 5) conforming to theformula and which is one of the possible species included in the groupdescribed by SEQ. ID. NO: 1, was shown to behave identically to aselected binding antagonist.

1. A peptide which is a high affinity antagonist of binding of ananti-integrin antibody comprising a peptide having the amino acidsequence of the formula: Asp Phe Xaa₁ Ser Xaa₂ Trp Xaa₃ Xaa₄ Xaa₅ Xaa₆Xaa₇ Xaa₈, wherein Xaa₂ is selected from Trp, Tyr and Phe, Xaa₃ isselected from Glu and Asp, Xaa₄ is selected Ile, Leu and Val and Xaa₁,Xaa₅ Xaa₆ Xaa₇ Xaa₈ are independently selected from any naturallyoccurring amino acid (SEQ. ID. No.1).
 2. A peptide according to claim 1of the formula: DFRSWWDLSGYR. (SEQ. ID. No.2)


3. A peptide according to claim 1 of the formula: DFRSWWDLEE. (SEQ. ID.No.5)


4. The use of a peptide of claim 1 as an immunogen to generateanti-integrin antibodies.
 5. An isolated anti-integrin antibody orantibody fragment which binds to a peptide of claim 1 such that theanti-integrin antibody is substantially prevented from binding tonaturally occurring biologically active ligands, other than the antibodyCNTO95.
 6. A pharmaceutical composition comprising an antibody of claim5 in combination with a pharmaceutically acceptable carrier.
 7. A methodof using an anti-integrin antibody of claim 5 to treat a diseasecharacterized by angiogenesis, wherein the angiogenesis-dependentdiseases is a disease selected from the group consisting of cancermetastasis, angioma, angiofibroma, diabetic retinopathy, prematureinfant's retinopathy, neovascular glaucoma, corneal disease induced byangiogenesis, involutional macula, macular degeneration, pterygium,retinal degeneration, retrolental fibroplasias, granular conjunctivitis,psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis,acne and arthritis.
 8. The method according to claim 7, in which saidmonoclonal antibody treats an inflammatory disease selected from thegroup consisting of rheumatoid arthritis, macular degeneration,psoriasis, diabetic retinopathy.
 9. The method according to claim 7, inwhich said monoclonal antibody treats an angiogenic skin disorderselected from the group consisting of psoriasis, venous ulcers, acne,rosacea, warts, eczema, hemangiomas, and lymphangiogenesis.
 10. Themethod according to claim 7, in which said monoclonal antibody treatsdisorder involving corneal or retinal neovascularization.
 11. A methodfor the selection of an integrin receptor binding ligand from a receptorbinding ligand library, which comprises screening the library forreceptor binding ligands which bind to a peptide of claim
 1. 12. Amethod of identifying antagonist ligands to a therapeutic monoclonalantibody, which method comprises synthesizing a collection of peptides,displaying the peptides on the surface of a phage to create a phagedisplay library of such peptides, and screening the peptide displaylibrary for peptides which bind to the therapeutic monoclonal antibodywith the desired characteristics.
 13. A method of claim 12, furthercomprising the steps of aligning the sequences of the binding peptides,designating the subregions of the binding surface in terms of types ofamino acid residue to be positioned in said subregion, and creating apeptide where the sequence and positional relationship of each residuein the sequence conforms to the designated subregions.